Emerging Role of Extracellular Vesicles in Embryo-Maternal Communication throughout Implantation Processes.

In ruminants, the establishment of proper conceptus-endometrial communication is essential for conceptus implantation and subsequent successful placentation. Accumulated evidence supports the idea that extracellular vesicles (EVs) present in uterine lumen are involved in conceptus-endometrial interactions during the preimplantation period. EVs make up a new field of intercellular communicators, which transport a variety of bioactive molecules, including soluble and membrane-bound proteins, lipids, DNA, and RNAs. EVs thus regulate gene expression and elicit biological effects including increased cell proliferation, migration, and adhesion in recipient cells. Uterine EVs are interactive and coordinate with ovarian progesterone (P4), trophectoderm-derived interferon tau (IFNT) and/or prostaglandins (PGs) in the physiological or pathological microenvironment. In this review, we will focus on intrauterine EVs in embryo-maternal interactions during the early stage of pregnancy, especially the implantation period in ruminant ungulates.

Can centrifugation force compromise the plasmatic membrane, acrosome and DNA integrity of goat spermatozoa? / A força de centrifugação pode comprometer a integridade de membrana plasmática, acrossomal e DNA de espermatozoides caprinos?

Protocols for cooling or freezing goat semen usually recommend centrifugation for seminal plasma removal. However, little is known about the effect of this process on goat sperm viability and functionality. The present study evaluated the effects of centrifugation force on the plasma membrane, acrosomes, and DNA integrity of goat semen. Four ejaculates from each of the four different Anglo Nubian male goats were used. Semen samples were obtained using artificial vagina, and immediately after collection, ejaculates were diluted using Ringer's sodium lactate solution and split into three groups: Control (CG, without centrifugation), G1 (centrifugation 600 x g/10 min), G2 (centrifugation 1200 x g/10 min). After centrifugation, seminal plasma was removed, the sperm pellets were resuspended using Tris-egg yolk extender (80 x 106 spermatozoa/mL) and the sperm morphology was analyzed. Samples were cooled at 5°C for 5, 24, 36, and 48 h and then sperm plasma membrane and acrosome integrity (PMAI, %) and sperm DNA fragmentation index (SDF, %) were evaluated at each time-point, using a flow cytometer. Additionally, sperm movement was determined using computer semen analysis (CASA) after 5, 24, and 48 h of refrigeration period. The semen centrifugation did not induce additional sperm morphology defect or reduction in sperm kinetics in the experimental groups. Differences were not observed (p > 0.05) in PMAI and SDF among different groups, in any of each time-point of the cooling process. In conclusion, centrifugation, even at high speeds, did not affect goat sperm integrity and functionality when submitted to refrigeration process. (AU)

A maior parte dos protocolos de refrigeração e criopreservação do sêmen caprino recomenda o uso de centrifugação para remoção do plasma seminal. No entanto, não existe consenso sobre o risco que esse tipo de processamento pode ocasionar à viabilidade espermática. Nesse contexto, o presente trabalho investigou os possíveis efeitos deletérios da centrifugação sobre a integridade estrutural e DNA de espermatozoides caprinos. Para a pesquisa foram selecionados quatro reprodutores para colheita de sêmen (n = 4 ejaculados/bode). Cada ejaculado foi fracionado em três alíquotas iguais, diluídas em ringer e divididas em três grupos: Controle (GC, não centrifugado), G1 (centrifugação a 600 g/10 minutos) e G2 (centrifugação a 1200 g/10 minutos). As amostras seminais por grupo foram diluídas em meio Tris gema respeitando-se a concentração final de 80 milhões de espermatozoides/mL e foram submetidas à avaliação de morfologia espermática. Todas as amostras foram acondicionadas a 5°C, sendo analisadas nos momentos 5, 24, 36 e 48 horas do processo de refrigeração por meio da avaliação da integridade de membrana plasmática e acrossomal (MPAI, %) e índice de fragmentação de DNA (IDF, %). Adicionalmente, a cinética espermática foi avaliada com o emprego de um sistema computadorizado de análise (CASA) nos momentos 5, 24 e 48 horas da refrigeração. A centrifugação não induziu a manifestação de defeitos morfológicos ou redução significativa da cinética de espermatozoides caprinos. Não foram observadas diferenças para a integridade de membrana plasmática e para o índice de fragmentação de DNA quando comparados, respectivamente, GC, G1 e G2 em cada um dos quatro momentos experimentais. Conclui-se que mesmo quando empregadas altas forças de rotação não ocorre lesão à ultraestrutura dos espermatozoides caprinos submetidos ao processo de refrigeração.(AU)

Abortos, malformações congênitas e falhas reprodutivas espontâneas em caprinos causados na intoxicação pelas folhas da catingueira, Poincianella pyramidalis (sin. Caesalpinia pyramidalis) / Spontaneous abortion, congenital malformation and reproductive losses in goats due to poisoning by the leaves of Poincianella pyramidalis (syn. Caesalpinia pyramidalis)

O objetivo deste estudo foi investigar os efeitos de folhas da espécie arbórea Poincianella pyramidalis (Tul.) L.P. Queiroz como causa espontânea de abortos, malformações e falhas reprodutivas em rebanhos caprinos no semiárido brasileiro. Foram realizadas investigações epidemiológicas, avaliações clínicas de caprinos acometidos por malformações e necropsias de fetos abortados em 12 propriedades localizadas nos municípios de Gurjão e Soledade, na Paraíba, e no município de Pau dos Ferros, Rio Grande do Norte. O estudo considerou apenas as propriedades que tinham basicamente a presença de P. pyramidalis entre as plantas teratogênicas. Na propriedade localizada no município de Pau dos Ferros foi verificada a ocorrência de grande número de abortos e o nascimento de cabritos malformados, acometendo 90% das cabras gestantes. Nas 12 propriedades pertencentes aos municípios de Gurjão e Soledade na Paraíba os casos espontâneos de mortalidade embrionária, abortos e malformações acometeram 257 cabras (41,1% das cabras dos rebanhos). A artrogripose foi a principal malformação congênita observada. Exames de necropsia e histopatológico de quatro fetos acompanhados das placentas não constataram a presença de agentes infecciosos. Os casos de perda embrionária, abortos e malformações foram concentrados predominantemente nas épocas do ano em que a pastagem estava escassa, mas havia disponibilidade de P. pyramidalis nas propriedades, devido à ocorrência das chuvas da pré-estação, que não permitiu a germinação de pasto, mas a rebrotação de P. pyramidalis. Os achados desse estudo comprovam que esta planta interfere consideravelmente na eficiência reprodutiva dos rebanhos caprinos do Semiárido.(AU)

The aim of this study was to investigate the effects of the leaves of the tree Poincianella pyramidalis (Tul.) L.P. Queiroz as a spontaneous cause of abortion, malformation and reproductive failure in goat herds in the Brazilian semiarid region. Epidemiological investigations, clinical evaluation of goats affected by malformation and necropsy of aborted fetuses was carried out on 12 farms located in the municipalities of Gurjão and Soledade, state of Paraíba, and on a farm located at the municipality of Pau dos Ferros, state of Rio Grande do Norte. The study considered only the farms where occurred predominantly P. pyramidalis among teratogenic plants. On the farm located in the municipality of Pau dos Ferros the occurrence of frequent abortion and birth of malformed goats, affecting 90% of pregnant goats, was observed. On the 12 farms belonging to the municipalities of Gurjão and Soledade, spontaneous cases of embryonic mortality, abortion and malformation occurred in 257 goats (41.1% of goats). Arthrogryposis was the main congenital malformation observed. Necropsy and histopathological exams of four fetuses and their placentas did not detect the presence of infectious agents. The cases of embryonic mortality, abortion and malformation were predominantly concentrated during the seasons of the year when pasture was scarce, but P. pyramidalis was available, due to the occurrence of pre-seasonal rains, which did not allow the pasture to germinate but P. pyramidalis to regrowth. The findings of this study confirm that this plant significantly interferes in the reproductive efficiency of semi-arid goat herds.(AU)

Óleo de peixe associado ao ácido ascórbico no diluidor para criopreservação de sêmen caprino / Fish oil associated to ascorbic acid in diluter for cryopreservation goat semen

O presente estudo teve como objetivo avaliar o efeito da inclusão de óleo de peixe associado ao ácido ascórbico no diluidor para criopreservação de sêmen caprino. Dois machos da raça Boer foram submetidos à coleta de sêmen pelo método de vagina artificial, sendo os ejaculados avaliados quanto aos aspectos físicos e morfológicos. Após avaliação, formou-se um pool, seguido do fracionamento em cinco grupos: G1 - diluidor citrato-gema e G2, G3, G4 e G5 - diluidor citrato-gema acrescido de 1,0; 2,0; 3,0 e 4,0% de óleo de peixe e 0,05% de ácido ascórbico, respectivamente. Após descongelamento, foram realizadas avaliações físicas do sêmen e os testes complementares de termorresistência lento (TTR), hiposmótico (HO), integridade acrossomal e compactação da cromatina espermática. Houve comportamento linear crescente (P<0,05) para motilidade pós-descongelamento. Não houve diferença (P>0,05) para vigor pós-descongelamento (2,00±0,24). No TTR não houve diferença (P>0,05) para motilidade e vigor espermáticos entre os tempos cinco e 180min, com médias inicial e final de 62,17±12,13 e 14,29±10,55 para motilidade e de 2,00±0,52 e 0,49±0,44 para vigor. Não houve diferença (P>0,05) para o HO, com porcentagem média de espermatozoides reativos de 23,5±5,96%. Houve comportamento linear crescente para acrossoma íntegro e decrescente para acrossoma irregular (P<0,05). Não houve diferença (P>0,05) na compactação da cromatina, com 97,06±1,17% de cromatina íntegra. A inclusão até 4% de óleo de peixe acrescido de ácido ascórbico no diluidor melhorou motilidade e integridade de acrossoma após a criopreservação.(AU)

The study aimed to evaluate the effect of fish oil inclusion associated with ascorbic acid in the thinner for goat semen cryopreservation. Two male Boers underwent semen collection through the artificial vagina method, ejaculates being then assessed for physical and morphological aspects. After evaluation, a pool was formed, followed by the split into five groups: G1 - yolk-citrate extender and G2, G3, G4 and G5 - yolk-citrate extender plus 1.0; 2.0; 3.0 and 4.0% fish oil and 0.05% ascorbic acid, respectively. After thawing, physical evaluations of semen were assessed and additional testing slow heat resistance (TTR), hiposmotic (HO), acrosome integrity and compression of sperm chromatin. There was linear increase (P<0.05) post-thaw motility. No difference was obtained for post-thaw vigor and there was no influence of the association of fish oil and ascorbic acid in TTR. Plasma membrane integrity, by hyposmotic test (HO), presented a mean of reactive spermatozoa of 23.5±5.96% (P>0.05). There was linear increase for intact acrosome and decreasing acrosome irregular (P<0.05). In the analysis of the chromatin compaction, approximately 3% of damages (P>0.05) were observed. The inclusion of 4% fish oil plus ascorbic acid in diluter improved motility and acrosome integrity after cryopreservation.(AU)

Role of lipids on elongation of the preimplantation conceptus in ruminants.

Elongation of the preimplantation conceptus is a prerequisite for successful pregnancy in ruminants and depends on histotroph secretion by the endometrium. Lipids are an essential component of the histotroph, and recent studies indicate that lipids have important roles in the elongation phase of conceptus development. The onset of elongation is marked by dynamic changes in the transcriptome of trophectoderm cells, which are associated with lipid metabolism. During elongation, the trophectoderm increases transcript expression of genes related to uptake, metabolism and de novo biosynthesis of fatty acids and prostaglandins. Expression of the gene PPARG increases substantially, and activation of the transcription factor PPARG by binding of lipid ligands appears to be crucial for the coordination of cell biology during elongation. Lipids accumulated in the epithelial cells of the endometrium during diestrus are likely the most important source of fatty acids for utilization by the conceptus and become available in the uterine lumen through exporting of exosomes, microvesicles, carrier proteins and lipoproteins. Targeting of uterine lipid metabolism and PPARG activity during preimplantation conceptus development through nutraceutical diets may be a good strategy to improve pregnancy survival and reproductive efficiency in ruminants.

The role of progesterone and conceptus-derived factors in uterine biology during early pregnancy in ruminants.

This review integrates established and new information on the role of progesterone, interferon tau (IFNT), and prostaglandins in uterine biology of ruminants. Establishment of pregnancy in ruminants encompasses growth of the posthatching blastocyst, elongation of the conceptus (embryo and extraembryonic membranes), and suppression of the endometrial luteolytic mechanism to maintain progesterone production by the ovary. Conceptus elongation involves exponential increases in length of the trophectoderm for pregnancy recognition signaling, implantation, and establishment of pregnancy. Pregnancy recognition signaling is accomplished by IFNT from the trophectoderm that has a paracrine antiluteolytic effect to inhibit upregulation of oxytocin receptors in the endometrial epithelia, thereby inhibiting production of luteolytic PGF2α pulses by the uterus. Survival and growth of the preimplantation blastocyst and elongating conceptus clearly requires embryotrophic factors (AA, carbohydrates, proteins, lipids, and other substances) in the uterine lumen. Individual, interactive, and coordinated actions of progesterone, IFNT, and prostaglandins regulate expression of elongation- and implantation-related genes in the endometrial epithelia that, in turn alter the uterine luminal histotroph and govern conceptus survival and growth. An increased knowledge of progesterone biology and conceptus-endometrial interactions is necessary to understand and elucidate the causes of pregnancy loss and provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency in ruminants.

The effect of nutrition and metabolic status on the development of follicles, oocytes and embryos in ruminants.

The impact of nutrition and energy reserves on the fertility of ruminants has been extensively described. However, the metabolic factors and the molecular mechanisms involved in the interactions between nutrition and ovarian function are still poorly understood. These factors could be hormonal (either reproductive and/or metabolic) and/or dietary and metabolic (glucose, amino acids and fatty acids). In this review, we briefly summarize the impact of those nutrients (fatty acids, glucose and amino acids) and metabolic hormones (insulin/IGF-I, growth hormone, T3/4, ghrelin, apelin and the adipokines (leptin, adiponectin and resistin)) implicated in the development of ovarian follicles, oocytes and embryos in ruminants. We then discuss the current hypotheses on the mechanisms of action of these factors on ovarian function. We particularly describe the role of some energy sensors including adenosine monophosphate-activated kinase and peroxisome proliferator-activated receptors in the ovarian cells.

In vitro production of small ruminant embryos: late improvements and further research.

Beyond the potential use of in vitro production of embryos (IVP) in breeding schemes, embryos are also required for the establishment of new biotechnologies such as cloning and transgenesis. Additionally, the knowledge of oocyte and embryo physiology acquired through IVP techniques may stimulate the further development of other techniques such as marker assisted and genomic selection of preimplantation embryos, and also benefit assisted procreation in human beings. Efficient in vitro embryo production is currently a major objective for livestock industries, including small ruminants. The heterogeneity of oocytes collected from growing follicles by laparoscopic ovum pick up or in ovaries of slaughtered females, remains an enormous challenge for IVM success, and still limits the rate of embryo development. In addition, the lower quality of the IVP embryos, compared with their in vivo-derived counterparts, translates into poor cryosurvival, which restricts the wider use of this promising technology. Therefore, many studies have been reported in an attempt to determine the most suitable conditions for IVM, IVF, and in vitro development to maximize embryo production rate and quality. This review aims to present the current panorama of IVP production in small ruminants, describing important steps for its success, reporting the recent advances and also the main obstacles identified for its improvement and dissemination.

Ovarian and placental morphology and endocrine functions in the pregnant giraffe (Giraffa camelopardalis).

Gross, histological and immunocytochemical examinations carried out on maternal and fetal reproductive tissues from two pregnant giraffes at an estimated 8 and 13.5 months of gestation (term=15 months) revealed a typically ruminant macrocotyledonary placenta with binucleate trophoblast cells scattered sparsely in the placentome where they stained intensely with a prolactin antiserum. Binucleate cells were present in greater numbers in the intercotyledonary allantochorion where they did not stain for prolactin whereas the uninucleate trophoblast still did. A single large corpus luteum of pregnancy and several small luteinised follicles were present in the maternal ovaries while the fetal ovaries at 13.5 months gestation showed an assortment of enlarging antral follicles and partially and completely lutenised follicles, the granulosa and luteal cells of which stained positively for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17,20 lyase, prolactin, progesterone receptor and androgen receptor, but negatively for aromatase. The uninucleate trophoblast of the placentome and intercotyledonary allantochorion, the epithelium of the maternal endometrial glands, the seminiferous epithelium in the fetal testis at 8 months of gestation and the zonae fasciculata and reticularis of the fetal adrenal at 13.5 months also stained positively for 3ß-HSD and negatively for aromatase. Endocrinologically, it appears that the giraffe placenta is more similar to that of the sheep than the cow with a placental lactogen as the likely driver of the considerable degree of luteinisation seen in both the maternal and the fetal ovaries.

The entotympanic in late fetal Artiodactyla (Mammalia).

The entotympanic is a neomorphic component of the bulla tympanica of placental mammals. Ontogenetically, its rostral component seems to be derived from the tubal cartilage, whereas its caudal component is normally connected with the sheath of the tympanohyal; the present study indicates additional sources of the caudal entotympanic. The entotympanics develop in late fetal or early postnatal life as cartilaginous structures, but in most taxa they ossifiy endochondrally as "os bullae". This skeletal element is absent only in a few placental orders, among them the Artiodactyla. Because it is present in their sister taxa within the Scrotifera, it is likely to be reduced secondarily in the even-toed mammals. The study of histological serial sections of late fetal stages of several artiodactyl species shows that vestigial cartilaginous homologues of the entotympanics are invariably present, contrary to statements in the literature. In a few perinatal stages even secondary ossifications or calcifications of the entotympanic cartilages can be observed. The tubal cartilage of artiodactyls also continues into an anterior tegmen tympani (new term) that forms the floor of the fossa muscularis major.

A review of poisonous plants that cause reproductive failure and malformations in the ruminants of Brazil.

The objective of this review is to provide a report on toxic plants causing reproductive problems in ruminants in Brazil. Aspidosperma pyrifolium causes abortion or stillbirth in goats, as well as most likely in sheep and cattle, in the semiarid regions of Northeastern Brazil. Intoxications by Ateleia glazioveana, Tetrapterys acutifolia and T. multiglandulosa result in abortion and neonatal mortality in cattle and sheep, and the same signs have been experimentally observed in goats. These three plants can also cause cardiac fibrosis and a nervous disease with spongiosis of the central nervous system. Other plants known to cause abortion include Enterolobium contortisiliquum, E. gummiferum, Stryphnodendron coriaceum, S. obovatum and S. fissuratum. These plants can also cause digestive signs and photosensitization. Abortions have been reported in animals intoxicated by nitrates and nitrites as well. Infertility, abortions and the birth of weak offspring have been reported in animals intoxicated by plants containing swainsonine, including Ipomoea spp., Turbina cordata and Sida carpinifolia. Trifolium subterraneum causes estrogenism in cattle. Mimosa tenuiflora and, most likely, M. ophthalmocentra cause malformations and embryonic mortality in goats, sheep and cattle in the semiarid regions of Northeastern Brazil.

From fetus to adult--an allometric analysis of the giraffe vertebral column.

As mammalian cervical vertebral count is almost always limited to seven, the vertebral column of the giraffe (Giraffa camelopardalis) provides an interesting study on scaling and adaptation to shape in light of these constraints. We have defined and described the growth rates of the lengths, widths, and heights of the vertebrae from fetal through neonatal life to maturity. We found that the disproportionate elongation of the cervical vertebrae is not a fetal process but occurs after birth, and that each cervical (C2-C7) vertebrae elongates at the same rate. C7 is able to specialize toward elongation as its function has been shifted to T1. We concluded that T1 is a transitional vertebra whose scaling exponent and length is between that of the cervical and thoracic series. Despite its transitional nature, T1 is still regarded as thoracic, as it possesses an articulating rib that attaches to the sternum. The other dimensions taken (width, height, and spinous process length) show that giraffe vertebral morphology exhibit adaptations to biomechanical strain, and we have underlined the importance of the thoracic spinous processes in supporting the head and neck.

Fetal programming of skeletal muscle development in ruminant animals.

Enhancing skeletal muscle growth is crucial for animal agriculture because skeletal muscle provides meat for human consumption. An increasing body of evidence shows that the level of maternal nutrition alters fetal skeletal muscle development, with long-term effects on offspring growth and performance. Fetal skeletal muscle development mainly involves myogenesis (i.e., muscle cell development), but also involves adipogenesis (i.e., adipocyte development) and fibrogenesis (i.e., fibroblast development). These tissues in fetal muscle are mainly derived from mesenchymal stem cells (MSC). Shifting the commitment of MSC from myogenesis to adipogenesis increases intramuscular fat (i.e., marbling), improving the quality grade of meats. Strong experimental evidence indicates that Wingless and Int (Wnt)/beta-catenin signaling regulates MSC differentiation. Upregulation of Wnt/beta-catenin promotes myogenesis, and downregulation enhances adipogenesis. A lack of nutrients in early to midgestation reduces the formation of secondary muscle fibers in ruminant animals. Nutrient deficiency during mid- to late gestation decreases the number of intramuscular adipocytes and muscle fiber sizes. Knowledge of this regulatory mechanism will allow the development of strategies to enhance muscle growth and marbling in offspring, especially in the setting of nutrient deficiency.

Genes involved in conceptus-endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches.

This review summarizes new knowledge on expression of genes and provides insights into approaches for study of conceptus-endometrial interactions in ruminants with emphasis on the peri-implantation stage of pregnancy. Conceptus-endometrial interactions in ruminants are complex and involve carefully orchestrated temporal and spatial alterations in gene expression regulated by hormones from the ovary and conceptus. Progesterone is the hormone of pregnancy and acts on the uterus to stimulate blastocyst survival, growth, and development. Inadequate progesterone levels or a delayed rise in progesterone is associated with pregnancy loss. The mononuclear trophectoderm cells of the elongating blastocyst synthesize and secrete interferon-tau (IFNT), the pregnancy recognition signal. Trophoblast giant binucleate cells begin to differentiate and produce hormones including chorionic somatomammotropin 1 (CSH1 or placental lactogen). A number of genes, induced or stimulated by progesterone, IFNT, and/or CSH1 in a cell-specific manner, are implicated in trophectoderm adhesion to the endometrial luminal epithelium and regulation of conceptus growth and differentiation. Transcriptional profiling experiments are beginning to unravel the complex dynamics of conceptus-endometrial interactions in cattle and sheep. Future experiments should incorporate physiological models of pregnancy loss and be complemented by metabolomic studies of uterine lumen contents to more completely define factors required for blastocyst survival, growth, and implantation. Both reduction and holistic approaches will be important to understand the multifactorial phenomenon of recurrent pregnancy loss and provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency in cattle and other domestic animals.

Pregnancy recognition and conceptus implantation in domestic ruminants: roles of progesterone, interferons and endogenous retroviruses.

The present review highlights new information on pregnancy recognition and conceptus development and implantation in sheep with respect to regulation by progesterone, interferons and endogenous retroviruses. After formation of the corpus luteum, progesterone acts on the endometrium and stimulates blastocyst growth and elongation to a filamentous conceptus (embryo/fetus and associated extra-embryonic membranes). The envelope of endogenous retroviruses related to Jaagsiekte sheep retroviruses appears to intrinsically regulate mononuclear trophectoderm cell proliferation and differentiation into trophoblast giant binucleate cells. The mononuclear trophectoderm cells of elongating sheep conceptuses secrete interferon-tau, which acts on the endometrium to prevent development of the luteolytic mechanism by inhibiting transcription of the gene for the oestrogen receptor alpha in the luminal and superficial ductal glandular epithelia. These actions prevent oestrogen-induced transcription of the oxytocin receptor gene and, therefore, oxytocin-induced luteolytic pulses of prostaglandin F2alpha. Progesterone down regulation of its receptors in luminal and glandular epithelia correlates temporally with a reduction in anti-adhesive mucin land induction of secreted galectin 15 (LGALSI5) and secreted phosphoprotein 1, which are proposed to regulate trophectoderm proliferation and adhesion. Interferon-c acts on the endometrial lumenal epithelium to induce WNT7A and to stimulate LGALS 15, cathepsin L and cystatin C, which are candidate regulators of conceptus development and implantation. The number of potential contributors to maternal recognition and establishment of pregnancy continues to grow and this highlights our limited appreciation of the complexity of the key molecules and signal transduction pathways that intersect during these key developmental processes. The goal of improving reproductive efficiency by preventing embryonic losses that occur during the peri-implantation period of pregnancy in domestic ruminants provides the challenge to increase our knowledge of endometrial function and conceptus development.

The influence of nuclear content on developmental competence of gaur x cattle hybrid in vitro fertilized and somatic cell nuclear transfer embryos.

In nondomestic and endangered species, the use of domestic animal oocytes as recipients for exotic donor nuclei causes the normal pattern of cytoplasmic inheritance to be disrupted, resulting in the production of nuclear-cytoplasmic hybrids. Evidence suggests that conflict between nuclear and cytoplasmic control elements leads to a disruption of normal cellular processes, including metabolic function and cell division. This study investigated the effects of nuclear-cytoplasmic interactions on the developmental potential of interspecies embryos produced by in vitro fertilization and somatic cell nuclear transfer: cattle x cattle, gaur x cattle, hybrid x cattle. Cattle control and hybrid embryos were examined for development to the blastocyst stage and blastocyst quality, as determined by cell number and allocation, apoptosis incidence, and expression patterns of mitochondria-related genes. These analyses demonstrated that a 100% gaur nucleus within a domestic cattle cytoplasmic environment was not properly capable of directing embryo development in the later preimplantation stages. Poor blastocyst development accompanied by developmental delay, decreased cell numbers, and aberrant apoptotic and related gene expression profiles, all signs of disrupted cellular processes associated with mitochondrial function, were observed. Developmental potential was improved when at least a portion of the nuclear genome corresponded to the inherited cytoplasm, indicating that recognition of cytoplasmic components by the nucleus is crucial for proper cellular function and embryo development. A better understanding of the influence of the cytoplasmic environment on embryonic processes is necessary before interspecies somatic cell nuclear transfer can be considered a viable alternative for endangered species conservation.

Production of blastocysts after intergeneric nuclear transfer of goral (Naemorhedus goral) somatic cells into bovine oocytes.

Interspecies cloning may be a useful method to help conserve endangered species and to study nuclear-cytoplasm interaction. The present study investigated in vitro development of goral (Naemorhedus goral) intergeneric nuclear transfer embryos produced by fusing goral fibroblasts with enucleated metaphase II (MII) bovine oocytes. After two to five passages, serum-starved or non-starved goral skin fibroblast cells were transferred into enucleated MII bovine oocytes. Couplets were electrically fused and chemically activated, and then cultured in either modified synthetic oviduct fluid (mSOF) or tissue culture medium-199 (TCM-199) supplemented with 10% FBS. Serum starvation of donor cells did not affect the fusion rate and or development to of cells to the two-cell stage, to more than 9-cells, or to morulae, regardless of culture medium. Three blastocysts from 202 fused embryos were obtained when embryos reconstructed with non- serum- starved donor cells were cultured in mSOF. However, no blastocysts were obtained when the embryos reconstructed with serum-starved donor cells were cultured in mSOF. The total cell number of goral intergeneric embryos averaged 130.3 (range 105-180). In conclusion, this study demonstrated that bovine oocytes can support blastocyst development after intergeneric SCNT with goral fibroblasts.

Obtaiment of Pudu (Pudu pudu) deer embryos by the somatic nuclear transfer technique

RESUMEN: Los objetivos de este trabajo fueron los de producir embriones de pudú, obtenidos por la transferencia de núcleos de fibroblastos de la oreja de pudú en ovocitos de un rumiante domésticos que es el bovino. Para posteriormente en un trabajo futuro proceder a la transferencia de embriones de pudú, al útero de hembras receptoras sincronizadas de otra especie. Se obtuvieron biopsias de 1 mm aproximadamente del borde externo de la orejas de dos ciervos pudu machos del jardín zoológico Buin-Zoo, Santiago de Chile. Las líneas celulares han sido establecidas y conservadas según los protocolos utilizados para las bovinos. Los ovocitos son obtenidos por punción del complejo cúmulos-ovocito (COC).desde ovarios de vacas recuperados del matadero. Cada ovocito es enucleado y fusionado con un fibroblasto aislado insertado bajo la zona pelúcida. La fusión de membranas celulares es obtenida por choques eléctricos. En cuanto a la cronología, observamos que al segundo día se forma una etapa de dos blastómeras, al tercer día mórulas de 8 a 16 células, y desde el cuarto día se ha diferenciado como blastocisto, el cuál al séptimo día termina por eclosionar de la zona pelúcida.La obtención de blastocistos embrionarios indica que es posible obtener embriones de pudú mediante clonaje heteroespecífico, aunque, el porcentaje de éxito obtenido es relativamente bajo. Queda aun por verificar la viabilidad de los embriones así obtenidos después de la transferencia in útero.

Analysis of fetal and maternal microvasculature in ruminant placentomes by corrosion casting.

Vascular corrosion casting is a useful tool for studying the vascular architecture of complex organs. The synepitheliochorial placenta of ruminants is composed of two closed blood circuits, a fetal and a maternal one. The microvasculature of each circuit has the shape of the corresponding cotyledon (villous trees) and caruncle (crypts). These two compartments interdigitate with each other in a complementary fashion. Understanding three-dimensional vascular arrangements is facilitated by scanning electron microscopy of vascular corrosion casts. Methods to be used in the generation of vascular casts from fetal and maternal placentomal blood vessels are described, with special emphasis on casting resins and corrosion using potassium hydroxide. The procedure of splitting larger casts following gelatin embedding and freezing is also presented.